Calcium ions play essential roles in signal transduction cascades for normal cellular functions but' also contribute to cell damage or death following ischemic injury and in specific pathological conditions of muscle and nerve. The molecular mechanisms responsible for mediating both the essential and deadly actions of calcium are not yet completely defined. Calpains (3.4.22.17), ubiquitous, intracellular, calcium-dependent proteolytic enzymes, undoubtedly mediate some the effects of calcium but their physiological regulation and function(s) are not yet known. These studies will identify appropriate cell culture systems to allow identification of the mechanisms that trigger activation of each isoform of calpain; micro-calpain and milli-calpain. The hypothesis we propose is that activation of each calpain is linked to a distinct calcium signal or source. Two excitable cell-types; neuronal-like (neuroblastoma N1E-115) and smooth muscle-like cells (DDT1 -MF2); and two non-excitable cell-types, the megakaryocyte-like erythroleukemia line (HEL) and bovine pulmonary endothelial cells (CLL-209) will be screened for their relative and absolute amounts of calpain and calpastatin. Each of these cell lines is relatively well characterized with regard to calcium regulation and calcium-linked signal pathways. Intracellular calcium homeostasis will be altered by 1) pharmacological means or 2) cells will be exposed to physiological effectors to determine which signal pathways stimulate calpain activation. Calpain activation will be measured by immunoblotting each calpain subunit with epitope-specific antibodies that distinguish the proenzyme from the activated enzyme and by measuring the degradation rates of calpain subunits. The experiments proposed will determine if proteolytic processing of either calpain is regulated by specific calcium signals or if calpain activation only occurs under conditions characterized by calcium overload. Whether calpains participate as mediators of cytotoxic responses that contribute to irreversible cell damage and death, or as mediators of calcium-linked signal transduction cascades required for cell growth or exocytosis, completion of these studies will provide the basis for future investigations of the significance of calpain in clinically important processes such as thrombosis, angiogenesis, tumor growth or neuro-degenerative diseases.